The present invention relates to a novel DNA coding for D-sorbitol dehydrogenase of a microorganism belonging to acetic acid bacteria including the genus Gluconobacter and the genus Acetobacter, an expression vector containing the said DNA, and recombinant organisms containing the said expression vector. Furthermore, the present invention relates to a process for producing recombinant D-sorbitol dehydrogenase protein and a process for producing L-sorbose by utilizing the said recombinant enzymes or recombinant organisms containing the said expression vector.
L-Sorbose is an important intermediate in the actual industrial process of vitamin C production, which is mainly practiced by Reichstein method (Helvetica Chimica Acta, 17: 311, 1934). In the process, the only microbial conversion is the L-sorbose production from D-sorbitol by Gluconobacter or Acetobacter strains. The conversion is considered to be carried out by NAD/NADP independent D-sorbitol dehydrogenase (SLDH). L-Sorbose is also a well known substrate in the art for microbiologically producing 2-keto-L-gulonic acid, which is a useful intermediate in the production of vitamin C.
It is known that there are NAD/NADP-independent D-sorbitol dehydrogenases which catalyze the oxidation of D-sorbitol to L-sorbose. One such D-sorbitol dehydrogenase was isolated and characterized from Gluconobacter suboxydans var xcex1 IFO 3254 (E. Shinagawa et al., Agric Biol. Chem., 46: 135-141, 1982), and found to consist of three subunits with the molecular weight of 63 kDa, 51 kDa, and 17 kDa; the largest subunit is dehydrogenase containing FAD as a cofactor, the second one is cytochrome c and the smallest one is a protein with unknown function; and shows its optimal pH at 4.5. Such SLDH was also purified and characterized from G. suboxydans ATCC 621 (KCTC 2111) (E-S Choi et al., FEMS Microbiol. Lett. 125:45-50, 1995) and found to consist of three subunits with the molecular weight of 75 kDa, 50 kDa, and 14 kDa; the large subunit is dehydrogenase containing pyrroloquinoline quinone (PQQ) as a cofactor, the second one is cytochrome c and the small one is a protein with unknown function. The inventors also purified and characterized the NAD/NADP-independent SLDH from G. suboxydans IFO 3255 (T. Hoshino et al., EP 728840); the SLDH consists of one kind of subunit with the molecular weight of 79.0 +/xe2x88x920.5 kDa and shows its optimal pH at 6 to 7 and shows dehydrogenase activity on mannitol and glycerol as well as on D-sorbitol.
Although several SLDHs have been purified, their genes have not been cloned yet. It is useful to clone the SLDH gene for efficient production of the SLDH enzyme and for constructing recombinant organism having enhanced SLDH activity to improve the production yield of L-sorbose. It is also useful to introduce the said SLDH gene into desired organisms, for example, Gluconobacter converting L-sorbose to 2-keto-L-gulonic acid for constructing recombinant microorganisms which directly produce 2-keto-L-gulonic acid from D-sorbitol.
The present invention provides a polynucleotide (i.e. nucleotide sequence or gene) coding for D-sorbitol dehydrogenase protein (SLDH) originating from a microorganism belonging to acetic acid bacteria including the genus Gluconobacter and the genus Acetobacter; a DNA molecule comprising said nucleotide sequence as well as a combination of the said DNA with a DNA comprising a nucleotide sequence of a protein functional in activating the said SLDH in vivo, expression vectors carrying the DNA comprising SLDH nucleotide sequence or the said combination of the DNAs; recombinant organisms carrying the expression vectors; a process for producing the recombinant SLDH; and a process for producing L-sorbose by utilizing the recombinant SLDH or the recombinant organisms.
Therefore this invention is directed to a polynucleotide comprising a nucleotide sequence which encodes a protein having the amino acid sequence from position 25 to position 740 of SEQ ID NO: 2, or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence of positions 25-740 of SEQ ID NO. 2, which protein has D-sorbitol dehydrogenase (SLDH) activity, i.e. converts D-sorbitol to L-sorbose. Such a polynucleotide preferably encodes a protein derived from an acetic acid bacterium, in particular an acetic acid bacterium belonging to the genus Gluconobacter or the genus Acetobacter. This invention is also directed to an expression vector comprising such a polynucleotide, especially one which is functional in a microorganism belonging to the genus Gluconobacter or the genus Acetobacter or which is an E. coli. This invention also includes a recombinant organism comprising such an expression vector, especially one which has the polynucleotide on its chromosomal DNA, and preferably one which is a Gluconobacter, Acetobacter, or an E. coli. 
This invention is also directed to a polynucleotide as described above consisting of a polynucleotide comprising a nucleotide sequence from position 644 to position 2791 of SEQ ID NO:1, or a polynucleotide comprising a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1, or polynucleotides capable of hybridizing to the above polynucleotides, and which encode proteins having D-sorbitol dehydrogenase protein activity.
This invention includes a polynucleotide which comprises a nucleotide sequence from position 192 to position 569 of SEQ ID NO: 1 and a polynucleotide which is capable of hybridizing to this polynucleotide and which encodes a protein which functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention (SLDH) in vivo, for example by helping to form and stabilize the SLDH when both proteins are present in an in vivo environment. This invention is also directed to an expression vector comprising such a polynucleotide, especially one which is functional in a microorganism belonging to the genus Gluconobacter or the genus Acetobacter or which is an E. coli. This invention also includes a recombinant organism comprising such an expression vector, especially one which has the polynucleotide on its chromosomal DNA, and preferably one which is a Gluconobacter, Acetobacter, or an E. coli. 
Also part of this invention is a polynucleotide comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of SEQ ID NO. 3 or a protein derived from the polypeptide by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO. 3, which protein functions to activate a D-sorbitol dehydrogenase protein of this invention (SLDH), in vivo (by this is meant that when the protein and SLDH are together in vivo, for example in a microorganism, the protein functions to form and stabilize the active SLDH which catalyzes the conversion of D-sorbitol to L-sorbose).
Another polynucleotide of this invention comprises the nucleotide sequence of SEQ ID NO. 1.
Any of the individual polynucleotides described above may be found in vectors of this invention, especially expression vectors. Similarly, any of the polynucleotides described above may be found in a recombinant organism (i.e. host cell) of this invention, either integrated into the host DNA, or on a vector such as an expression vector of this invention. Preferred vectors are those which function in the preferred recombinant organisms which are members of the genus Acetobacter, the genus Gluconobacter, or are E. colis. 
Also part of this invention are polynucleotides which combine the nucleotide sequences of other polynucleotides of this invention. These sequences may be in tandem (i.e. end to end), or may be separated by other coding or noncoding sequences.
One such polynucleotide comprises a nucleotide sequence which encodes a protein having the amino acid sequence from position 25 to position 740 of SEQ ID NO: 2 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence (i.e. position 25-740 of SEQ ID NO. 2) which protein has D-sorbitol dehydrogenase activity and a nucleotide sequence from position 192 to position 569 of SEQ ID NO: 1 or a polynucleotide which is capable of hybridizing to this polynucleotide and which encodes a protein which functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Another such polynucleotide comprises a nucleotide sequence which encodes a protein having the amino acid sequence from position 25 to position 740 of SEQ ID NO: 2, or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence (i.e. position 25-740 of SEQ ID NO. 2) which protein has D-sorbitol dehydrogenase activity; and a nucleotide sequence which encodes a polypeptide having the amino acid sequence of SEQ ID NO: 3; or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence (i.e. SEQ ID NO: 3), which protein functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Another such polynucleotide comprises a nucleotide sequence from position 644 to position 2791 of SEQ ID NO: 1 or a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1, or polynucleotides capable of hybridizing to the above nucleotide sequences and encoding a D-sorbitol dehydrogenase protein of this. invention; and a nucleotide sequence from position 192 to position 569 of SEQ ID NO: 1 or a polynucleotide which is capable of hybridizing to that polynucleotide and, which encodes a protein which functions to activate the D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention, in vivo.
Yet another polynucleotide comprises a nucleotide sequence from position 644 to position 2791 of SEQ ID NO: 1 or a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1, or polynucleotides capable of hybridyzing to these nucleotide sequences and which encode a D-sorbitol dehydrogenase protein of this invention, and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of SEQ ID NO. 3 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence (i.e. SEQ ID NO. 3) which protein functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention, in vivo.
This invention includes a polynucleotide which comprises a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1 and a polynucleotide comprising a nucleotide sequence from position 192 to position 569 of SEQ ID NO. 1.
The polynucleotides of this invention are preferably obtained from Gluconobacter or Acetobacter organisms as noted above.
This invention is also. directed to recombinant organisms which are host cells for the polynucleotides of this invention, and preferably are capable of expressing these polynucleotides. The polynucleotide may be on a vector of any type or integrated into the recombinant organism""s own DNA. In the case of recombinant organisms which contain more than one polynucleotide of this invention, the polynucleotides may be on a single vector, may be on separate vectors, or may be integrated into the DNA of the recombinant organism. Within one host cell containing multiple polynucleotides, one or more polynucleotides may be integrated into the host. DNA while one or more are on vectors. Thus this invention contemplates a vector, especially an expression vector, containing any individual polynucleotide of this invention, and also a recombinant organism containing any individual polynucleotide of this invention. It is preferred that the recombinant organism contain two polynucleotides, one of which encodes a protein of this invention which has D-sorbitol dehydrogenase protein activity, and the other of which encodes a protein of this invention which functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Preferred vectors are those vectors which are designed to function in microorganisms which are members of the genera Gluconobacter or Acetobacter, or in E. coli. Similarly, preferred recombinant organisms as host cells of this invention are members of the genera Gluconobacter or Acetobacter, or are E. coli. 
Thus part of this invention is recombinant organism which contains both a polynucleotide comprising a nucleotide sequence which encodes a protein having the amino acid sequence from position 25 to position 740 of SEQ ID NO: 2 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence (i.e. 25-740 of SEQ ID NO.2) which protein has D-sorbitol dehydrogenase activity, and contains a polynucleotide comprises a nucleotide sequence from position, 192 to position 569 of SEQ ID NO: 1 or a polynucleotide which is capable of hybridizing to that polynucleotide and which encodes a protein which functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Also part of this invention is a recombinant organism which contains both a polynucleotide comprising a nucleotide sequence which encodes a protein having the amino acid sequence from position 25 to position 740 of SEQ ID NO: 2 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence (i.e. 25-740 of SEQ ID NO. 2), which protein has D-sorbitol dehydrogenase activity; and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of SEQ ID NO. 3 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in SEQ ID NO. 3 which protein functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Another recombinant organism of this invention contains both a polynucleotide comprising a nucleotide sequence from position 644 to position 2791 of SEQ ID NO: 1 or a polynucleotide capable of hybridizing to this polynucleotide and encoding a protein having D-sorbitol dehydrogenase activity; or a polynucleotide comprising a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1 or a polynucleotide capable of hybridizing to this polynucleotide and encodes a protein having D-sorbitol dehydrogenase activity; and a polynucleotide comprising a nucleotide sequence from position 192 to position 569 of SEQ ID NO: 1 or a polynucleotide which is capable of hybridizing to this polynucleotide and encoding a protein which functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Yet another recombinant organism of this invention contains both a polynucleotide comprising a nucleotide sequence from position 644 to position 2791 of SEQ ID NO: 1 or a polynucleotide capable of hybridizing to this polynucleotide and encoding a protein having D-sorbitol dehydrogenase activity; or a polynucleotide comprising a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1 or a polynucleotide capable of hybridizing to this polynucleotide and encoding a protein having D-sorbitol dehydrogenase activity, and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of SEQ. ID, NO. 3 or a protein derived from this protein by substitution, deletion, insertion or addition of one or more amino acids in SEQ ID NO. 3, which protein functions to activated a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Another recombinant organism of this invention contains both a polynucleotide comprising a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1 and a polynucleotide comprising a nucleotide sequence from position 192 to position 569 of SEQ ID NO. 1. A preferred such recombinant organism has these polynucleotides in its chromosomal DNA. It is also preferable that the recombinant organism belongs to the genus Gluconobacter, the genus Acetobacter, or is an E. coli. 
Also part of this invention are expression vectors containing one or more of the polynucleotides of this invention. Expression vectors are well known in the art and are described below. Preferred expression vectors are designed to function in Gluconobacter or Acetobacter organisms, or in E. coli. An expression vector of this invention may contain more than one polynucleotide of this invention which may be in tandem. (end-to-end) or may be separated within the vector by other DNA sequences. It is preferred that the vector contain two polynucleotides, one of which encodes a protein of this invention which has D-sorbitol dehydrogenase activity, and the other of which encodes a protein of this invention which functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
A particular expression vector of this invention contains a polynucleotide comprising a nucleotide sequence which encodes a protein having the amino acid sequence from position 25 to position 740 of SEQ ID NO: 2 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence (i.e. 25-740 of SEQ ID NO. 2) which protein has D-sorbitol dehydrogenase activity, and a polynucleotide comprising a nucleotide sequence from position 192 to position 569 of SEQ. ID NO: 1 or a polynucleotide which is capable of hybridizing to that polynucleotide and which encodes a protein which functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Another expression vector of this invention contains a polynucleotide comprising a nucleotide sequence which encodes a protein having the amino acid sequence from position 25 to position 740 of SEQ ID NO: 2 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence (i.e. 25-740 of SEQ ID NO. 2) which protein has D-sorbitol dehydrogenase. activity, and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of SEQ ID NO. 3 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in SEQ ID NO. 3 which protein functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Yet another expression vector of this invention contains a polynucleotide. comprising a nucleotide sequence from position 644 to position 2791 of SEQ ID NO: 1 or a polynucleotide comprising a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1 or polynucleotides capable of hybridizing to the above polynucleotides and which encode proteins having D-sorbitol dehydrogenase activity, and a polynucleotide comprising a nucleotide sequence from position 192 to position 569 of SEQ ID NO: 1 or a polynucleotide which is capable of hybridizing to that polynucleotide and which encodes a protein which functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
Yet another expression vector of this invention contains a polynucleotide comprising a nucleotide sequence from position 644 to position 2791 of SEQ ID NO: 1 or a polynucleotide comprising a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1 or polynucleotides capable of hybridizing to the above polynucleotides and which encode proteins having D-sorbitol dehydrogenase activity,and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of SEQ ID NO. 3 or a protein derived from that protein by substitution, deletion, insertion or addition of one or more amino acids in SEQ ID NO. 3 which protein functions to activate a D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention in vivo.
This invention also includes an expression vector which contains a polynucleotide comprising a nucleotide sequence from position 572 to position 2791 of SEQ ID NO. 1 and a polynucleotide comprising a nucleotide sequence from position 192 to position 569 of SEQ ID NO.
Another particular expression vector of this invention contains a polynucleotide comprising a nucleotide sequence which encodes a protein having the amino acid sequence from position 25 to position 740 of SEQ ID NO. 2, or a protein derived from that protein substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence of positions 25-740 of SEQ ID NO. 2, which protein has D-sorbitol dehydrogenase activity. A preferred such vector is functional in a microorganism belonging to the genus Gluconobacter, the genus Acetobacter, or E. coli. 
Also part of this invention is a process for producing recombinant D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention by cultivating any of the recombinant organisms of this invention as described specifically above, especially the recombinant organisms which contain two polynucleotides of this invention one encoding recombinant D-sorbitol dehydrogenase and the other encoding a protein which functions to activate the D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention(in vivo), in an appropriate medium and recovering the said recombinant D-sorbitol from the culture. Accordingly, part of this invention is a recombinant D-sorbitol dehydrogenase protein produced by this process. A preferred such recombinant D-sorbitol dehydrogenase is immobilized on a solid carrier for solid phase enzymatic reaction.
Another process of this invention is a process for producing L-sorbose which comprises converting D-sorbitol into L-sorbose with the aid of the recombinant D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention.
Yet another process of this invention is a process for producing L-sorbose which comprises converting D-sorbitol into L-sorbose by fermentation of any of the recombinant organisms of this invention as described specifically above especially the recombinant organisms which contain two polynucleotides of this invention one encoding recombinant D-sorbitol dehydrogenase protein and the other encoding a protein which functions to activate the D-sorbitol dehydrogenase protein encoded by a polynucleotide of this invention (in vivo), in an appropriate medium.